Cultivation soil for leguminous plants and use thereof, cultivation kit for leguminous plants, cultivation method for leguminous plants, and seedling of leguminous plants with cultivation soil

Abstract

The invention provides culture soil for leguminous plants. A cultivation kit for a leguminous plant using the culture soil, a method for cultivating a leguminous plant, and a seedling of a leguminous plant with the culture soil; the invention also discloses application of the culture soil in cultivation of leguminous plants. The culture soil for leguminous plants comprises a root endophyte-based plant symbiotic bacterium and a soil, wherein the root endophyte-based plant symbiotic bacterium comprises at least one type of bacterium selected from the group consisting of a bacterium belonging to the genus Cladophialophora, a bacterium belonging to the genus Exophiala, and a bacterium belonging to the genus Veronaeopsis.

Description

Technical Field

[0001] The present invention relates to a culture soil for leguminous plants and its uses, a cultivation kit for leguminous plants, a cultivation method of leguminous plants, and seedlings of leguminous plants with the culture soil. Background Art

[0002] According to the Food and Agriculture Organization of the United Nations, the arable land area where crops can be produced smoothly is about 40% of the total world arable land area. Currently, about 5 million hectares (ha) of arable land deteriorates further every year. If new measures related to the protection of arable land are not taken, it is expected that by 2050, the per capita arable land in the world will be reduced to 1 / 4 of the arable land level in 1960. Therefore, it is necessary to convert from the conventional farming method that emphasizes productivity to sustainable agriculture that emphasizes environmental protection.

[0003] In the conventional farming method that emphasizes productivity, sometimes only a specific type of crop is cultivated in a specific arable land. However, continuously cultivating the same crop in the same place promotes the proliferation of certain specific types of pathogens (for example, pests or pathogenic bacteria, etc.), the balance of the ecosystem is disrupted, and then the production of crops is damaged. This is because most of the pathogens are host-specific to crops. Therefore, as a method for solving this problem, a cultivation method using root endophytic fungi is known. Root endophytic fungi (Dark-Septate Endophyte; DSE) refer to useful microorganisms that form mycorrhizae with plants and parasitize inside plant roots and are in a symbiotic relationship with plants.

[0004] Patent Document 1 describes the soybean growth promotion effect of Bradyrhizobium japonicum classified in the genus Bradyrhizobium in a low-temperature environment. Patent Document 2 describes that by inoculating endophytes, namely Veronaeopsis simplex Y34, K45, or CBS strains, favorable characteristics for stabilizing the growth of crops can be imparted. Patent Document 3 describes that by inoculating the endophyte Veronaeopsis simplex Y34 strain, there is an effect of inhibiting the absorption of radioactive cesium in tomatoes. Patent Document 4 describes that by inoculating Azospirillum brasilense NI-10 strain and rhizobia, there are effects of promoting the growth of leguminous plants and increasing the yield. Patent Document 5 describes that by inoculating the endophyte Stenotrophomonas sp. MYK101 strain in leguminous plants, there are effects of promoting growth and increasing the yield.

[0005] Non-Patent Document 1 describes a cultivation method in which a fungus, Cladophialophora chaetospira SK51, is symbiotically associated with the seedlings of strawberries affected by Fusarium wilt induced by a fungus of the genus Fusarium. Prior Art Documents Patent Documents

[0006] Patent Document 1: Japanese Unexamined Patent Application Publication No. 2021-090395 Patent Document 2: Japanese Unexamined Patent Application Publication No. 2021-052740 Patent Document 3: Japanese Unexamined Patent Application Publication No. 2016-054711 Patent Document 4: Japanese Unexamined Patent Application Publication No. 08-109109 Patent Document 5: Japanese Unexamined Patent Application Publication No. 2015-027995

[0007] Non-Patent Document 1: Wiwiek Harsonowati et al., The Effectiveness of a Dark Septate Endophytic Fungus, Cladophialophora chaetospira SK51, to Mitigate Strawberry Fusarium Wilt Disease and With Growth Promotion Activities, Front. Microbiol., 2020. Apr. 15, Vol. 11, Art. 585 Summary of the Invention

[0008] As described above, various studies have been conducted on promoting plant growth using symbiotic bacteria, but further technologies are desired for promoting the growth of leguminous plants.

[0009] The subject of the present invention is to provide: a culture soil for leguminous plants capable of promoting the growth of leguminous plants and its use, a cultivation kit for leguminous plants, a cultivation method for leguminous plants, and seedlings of leguminous plants with culture soil.

[0010] Specific methods for solving the above problems include the following. <1> A culture soil for leguminous plants, which contains a root endophytic bacterial plant symbiotic bacterium and soil, and the root endophytic bacterial plant symbiotic bacterium contains at least one bacterium selected from the group consisting of bacteria of the genus Cladophialophora, bacteria of the genus Exophiala, and bacteria of the genus Veronaeopsis. <2>The culture soil for leguminous plants according to <1> above further contains rhizobia.< / <3>The culture soil for leguminous plants of plants according to <1> or <2> above, wherein the fungus of the genus Cladophialophora is Cladophialophora chaetospira.< / <4>The culture soil for leguminous plants according to any one of <1> to <3> above, wherein the root endophytic plant symbiont is a fungus of the genus Veronaeopsis.< / <5>The culture soil for leguminous plants according to <4> above, wherein the pH of the soil is 4 or more and less than 6.< / <6>The culture soil for leguminous plants according to any one of <1> to <3> above, wherein the root endophytic plant symbiont is a fungus of the genus Cladophialophora.< / <7>The culture soil for leguminous plants according to <6> above, wherein the pH of the soil is 6 or more and 7 or less.< / <8>A cultivation kit for leguminous plants, comprising: the culture soil for leguminous plants according to any one of <1> to <7> above, and a leguminous plant body.< / <9>A cultivation method for leguminous plants, comprising: cultivating leguminous plants using the culture soil for leguminous plants according to any one of <1> to <7> above.< / <10>The cultivation method for leguminous plants according to <9> above, wherein in the cultivation, the root endophytic plant symbiont and rhizobia are mixed with the plant body at the same time to cultivate the leguminous plants.< / <11>A seedling of a leguminous plant with culture soil, comprising: the culture soil for leguminous plants according to any one of <1> to <7> above, and a seedling of a leguminous plant.< / <12>The use of the culture soil for leguminous plants according to any one of <1> to <7> above in the cultivation of leguminous plants.< / Advantages of the Invention

[0011] According to the present invention, it is possible to provide: a culture soil for leguminous plants capable of promoting the growth of leguminous plants and its use, a cultivation kit for leguminous plants, a cultivation method for leguminous plants, and a seedling of a leguminous plant with culture soil.< / Description of the Drawings

[0012] Figure 1A It is a photograph showing the growth state of the whole plant of soybean seedlings on the 14th day after starting cultivation by mixing the root endophytic plant symbiont with soybean seedlings in Examples 1, 2, 3 and Comparative Example 1. Figure 1B It is a graph related to the dry mass of each of the (B) stem - leaf region and the root region of soybean seedlings on the 14th day of cultivation starting from mixing the root - endophytic plant symbiotic bacteria with soybean seedlings in Examples 1, 2, 3 and Comparative Example 1. Figure 2 It is a graph related to the dry mass of soybeans harvested on the 140th day of cultivation starting from mixing the root - endophytic plant symbiotic bacteria with soybean seedlings in Examples 1, 2, 3 and Comparative Example 1. Figure 3 It is a photograph showing the state of root nodules formed on the roots of the plant body on the 40th day of cultivation starting from mixing the root - endophytic plant symbiotic bacteria with soybean seedlings in Examples 1, 2, 3 and Comparative Example 1. Figure 4 It is a graph related to the amount of phosphorus absorbed in soybean plant bodies on the 0th day, 100th day, and 140th day of cultivation starting from mixing the root - endophytic plant symbiotic bacteria with soybean seedlings in Examples 1, 2, 3 and Comparative Example 1. Figure 5A It is a graph related to the (A) number of leaves in soybean plant bodies on the 20th day of cultivation starting from mixing the root - endophytic plant symbiotic bacteria with soybean seedlings in Examples 4, 5 and Comparative Example 2. Figure 5B It is a graph related to the (B) dry mass of soybean plant bodies on the 20th day of cultivation starting from mixing the root - endophytic plant symbiotic bacteria with soybean seedlings in Examples 4, 5 and Comparative Example 2. Figure 5C It is a photograph showing the (C) growth state of seedlings in soybean plant bodies on the 20th day of cultivation starting from mixing the root - endophytic plant symbiotic bacteria with soybean seedlings in Examples 4, 5 and Comparative Example 2. Figure 6A It is a graph related to the (A) number of roots in soybean plant bodies on the 27th day of cultivation in Examples 6 and Comparative Example 3. Figure 6B It is a graph related to the (B) number of leaves in soybean plant bodies on the 27th day of cultivation in Examples 6 and Comparative Example 3. Figure 7A It is a graph related to the (A) number of roots in soybean plant bodies on the 20th day of cultivation in Examples 7 and Comparative Example 4. Figure 7B It is a graph related to the (B) number of leaves in soybean plant bodies on the 20th day of cultivation in Examples 7 and Comparative Example 4. Detailed implementation mode

[0013] Hereinafter, an embodiment of the present invention will be described in detail. However, the present invention is not limited to the following embodiments. In the following description, its components (including element steps, etc.) are non-essential unless otherwise specifically stated. The same applies to numerical values and their ranges, which do not limit the present invention. In the present invention, the term "process" includes, in addition to a process independent of other processes, a process that cannot be clearly distinguished from other processes but can achieve the purpose of the process. In the present invention, the numerical ranges represented by "~" respectively include the numerical values described before and after "~" as the lower limit value and the upper limit value. In the stepwise numerical ranges described in the present invention, the upper limit value or the lower limit value described in one numerical range can be replaced by the upper limit value or the lower limit value of other stepwise numerical ranges. In addition, in the numerical ranges described in this specification, the upper limit value or the lower limit value of the numerical range can be replaced by the value shown in the examples. In the present invention, the content rate of each component in the composition, when there are multiple substances belonging to each component in the composition, represents the total content rate of the multiple substances present in the composition unless otherwise specified. In the present invention, even when an element is expressed in the singular form, unless otherwise specifically stated, the existence of the plural is not excluded as long as there is no technical contradiction.

[0014] Cultivation Soil for Leguminous Plants The cultivation soil for leguminous plants of the present invention is a cultivation soil for leguminous plants containing root endophytic plant symbiotic bacteria and soil, and the root endophytic plant symbiotic bacteria contain at least one kind of bacteria selected from the genus Cladophialophora, the genus Exophiala, and the genus Veronaeopsis. The cultivation soil for leguminous plants of the present invention can promote the growth of leguminous plants by having the above composition. Its mechanism of action is not necessarily clear, but it can be speculated as follows. The root endophytic plant symbiotic bacteria containing at least one kind of bacteria selected from the genus Cladophialophora, the genus Exophiala, and the genus Veronaeopsis contained in the cultivation soil extend hyphae in a mesh-like manner around the roots of leguminous plants. In addition, if the bacteria attach to the surface of the roots of leguminous plants, an attachment organ is formed, which invades and fixes inside the cells of the roots of leguminous plants, that is, the symbiosis between the bacteria and the leguminous plants is achieved. If symbiosis is achieved, amino acids, proteins, etc. that cannot be effectively utilized as nutrients by leguminous plants before symbiosis can also be easily provided to plants by fungi. Thus, it is considered that leguminous plants are more likely to absorb nutrients such as nitrogen and phosphorus and are more likely to promote growth compared to before symbiosis.

[0015] In the present invention, the growth promotion of leguminous plants can be confirmed by the following method. Compared with the cultivation of leguminous plants using a culture soil that does not contain plant symbiotic bacteria of the root endophytic bacterial system, the dry mass of the above-ground parts such as the fruits, leaves, and stems of the cultivated leguminous plants increases, or the dry mass of the roots of the cultivated leguminous plants increases.

[0016] There is no particular limitation on the type of leguminous plants cultivated in the culture soil for leguminous plants of the present invention, and well-known leguminous plants such as soybeans, peas, broad beans, and adzuki beans can be applied. Among the above leguminous plants, the culture soil for leguminous plants of the present invention is particularly excellent in promoting the growth of soybeans and adzuki beans.

[0017] <Root endophytic bacterial system plant symbiotic bacteria> The root endophytic bacterial system plant symbiotic bacteria include at least one kind of bacteria selected from the genus Cladophialophora, the genus Exophiala, and the genus Veronaeopsis. The root endophytic bacterial system plant symbiotic bacteria can be used alone or in combination of two or more.

[0018] From the viewpoint of further promoting the growth of leguminous plants, the root endophytic bacterial system plant symbiotic bacteria preferably include at least one kind of bacteria selected from the genus Cladophialophora and the genus Veronaeopsis, and more preferably include one of the genus Cladophialophora or the genus Veronaeopsis.

[0019] The root endophytic bacterial system plant symbiotic bacteria refer to microorganisms that form mycorrhizae with plants and use the inside of plant roots as a parasitic site and are in a symbiotic relationship with plants.

[0020] Examples of the fungus of the genus Cladophialophora involved in the present invention include Cladophialophora chaetospira, Cladophialophora arxii, Cladophialophora tortuosa, Cladophialophora floridana, Cladophialophora psammophila, Cladophialophora boppii, Cladophialophora hachijoensis, Cladophialophora carrionii, Cladophialophora tumbae, and Cladophialophora tumulicola. Among them, from the viewpoint of further promoting the growth of leguminous plants, Cladophialophora chaetospira is preferred as the fungus of the genus Cladophialophora. The fungus of the genus Cladophialophora can be used alone or in combination of two or more kinds.

[0021] As Cladophialophora chaetospira, Cladophialophora chaetospira SK51 (hereinafter also referred to as SK51 or SK51 strain) deposited under accession number NITE BP-03539 or its mutant strain is preferred. The SK51 strain is deposited at the Patent Microorganisms Depositary, National Institute of Technology and Evaluation, with an address at 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan (date of deposit: November 07, 2022).

[0022] Examples of the method for introducing mutations include methods of treatment with chemical substances such as nitroso compounds (e.g., nitrosamines or nitrosoguanidines) or alkylating agents (e.g., EMS; ethyl methanesulfonate), ultraviolet irradiation, or radiation irradiation, etc., but are not limited thereto. Whether the obtained mutant strain exhibits the same or higher effect as the SK51 strain is detected by the following method: evaluating the effect of the obtained mutant strain on promoting the growth of leguminous plants and comparing the measurement results of this effect with those of the SK51 strain.

[0023] As fungi of the genus Exophiala, known species such as Exophiala pisciphila and their mutant strains, as well as Exophiala sp. SK47, etc. can be cited. Among them, as fungi of the genus Exophiala, from the viewpoint of further promoting the growth of leguminous plants, Exophiala sp. SK47 is preferred. Fungi of the genus Exophiala can be used alone as one kind, or two or more kinds can be used in combination.

[0024] As fungi of the genus Veronaeopsis, for example, Veronaeopsis simplex and its mutant strains, etc. can be cited. Among them, as fungi of the genus Veronaeopsis, from the viewpoint of further promoting the growth of leguminous plants, Veronaeopsis simplex is preferred. Fungi of the genus Veronaeopsis can be used alone as one kind, or two or more kinds can be used in combination.

[0025] The method for manufacturing the culture soil for leguminous plants of the present invention is not particularly limited, and the manufacturing method of the culture soil containing known fungi can be applied. The method for manufacturing the culture soil for leguminous plants of the present invention, for example, can mix and culture a culture solution containing root endophytic plant symbiotic bacteria (for example, 1×10 5 hyphal fragments / ml to 1×10 6 hyphal fragments / ml) with a culture material (for example, a mixture of wheat bran, rice bran, leaf mold, and sterilized water), and then manufacture it by further mixing the culture material with soil after culturing (for example, culturing in a chamber for 3 to 4 weeks). At this time, from the viewpoint of further promoting the growth of leguminous plants, the content of the culture material containing root endophytic plant symbiotic bacteria is preferably 5% to 10% by mass based on the total amount of the culture soil for leguminous plants.

[0026] The total number of fungi of the root endophytic plant symbiotic bacteria is not particularly limited. From the viewpoint of further promoting the growth of leguminous plants, it is preferably 1×10 3 hyphal fragments / g or more based on the culture soil for leguminous plants. It should be noted that since there is a symbiotic relationship between leguminous plants and root endophytic plant symbiotic bacteria, the number of fungi of the root endophytic plant symbiotic bacteria in the culture soil increases as the cultivation time of leguminous plants passes. The above-mentioned number of fungi is the number of fungi at the time of starting the cultivation of leguminous plants in the culture soil for leguminous plants (for example, at the time of sowing plant seeds in the culture soil, or at the time of transplanting and planting the 3-leaf stage seedlings cultivated separately in the culture soil, etc.).

[0027] The number of fungal strains of the root endophytic plant symbiotic bacteria can be measured by the following method: The fungal strains are plated on a 50% by mass CMMY agar medium and then cultured at 23 °C for 7 days.

[0028] The morphological form of the root endophytic plant symbiotic bacteria in the culture soil can be any form in the life cycle of the fungal strain. The form of the fungal strain can be, for example, a mycelium or a sporophyte.

[0029] The culture soil for leguminous plants can also contain fungal strains of the genus Cladophialophora, fungal strains of the genus Exophiala, and other root endophytic plant symbiotic bacteria other than the fungal strains of the genus Veronaeopsis, within the range where the effects of the present invention can be exerted. As other root endophytic plant symbiotic bacteria, for example, Meliniomyces variabilis, Phialocephala fortinii, etc. can be cited.

[0030] The culture soil for leguminous plants can also contain other microorganisms other than the root endophytic plant symbiotic bacteria, within the range where the effects of the present invention can be exerted. As other microorganisms, for example, in addition to the rhizobia described later, agrobacterium pusense (for example, Rhizobium sp. Y9, etc.), bacteria of the genus Pseudomonas, bacteria of the genus Paenibacillus, bacteria of the genus Stenotrophomonas, bacteria of the genus Delftia, etc. can be cited.

[0031] <Soil> The soil can be, for example, any one of organic cultivation soil, conventional cultivation soil (i.e., inorganic cultivation soil), and their mixed soil. The organic cultivation soil refers to soil that does not contain pesticides and chemical fertilizers. The conventional cultivation soil (i.e., inorganic cultivation soil) refers to soil that contains pesticides and / or chemical fertilizers.

[0032] From the viewpoint of further exerting the effects of the present invention, the pH of the soil is preferably 3 or more and 7 or less. The pH of the soil can be, for example, 3 or more and less than 4, 4 or more and less than 6, or 6 or more and 7 or less.

[0033] The pH of the soil can be measured by the following method. Specifically, the soil and distilled water are mixed at a ratio of 1:2.5 (preferably 1:5 when using soil with a high organic matter content), stirred with a reciprocating shaker for more than 1 hour, and then at 23°C ± 2°C, the pH of the suspension is measured by the glass electrode method, and the obtained value is taken as the pH of the soil.

[0034] For example, when the root endophytic plant symbiotic bacterium is a bacterium of the genus Veronaeopsis, the soil is preferably pH 4 or more and less than 6 from the viewpoint of further promoting the growth of leguminous plants.

[0035] For example, when the root endophytic plant symbiotic bacterium is a bacterium of the genus Cladophialophora, the soil is preferably pH 6 or more and 7 or less from the viewpoint of further promoting the growth of leguminous plants.

[0036] <Rhizobia> The culture soil for leguminous plants preferably further contains rhizobia from the viewpoint of further promoting the growth of leguminous plants. Rhizobia refer to bacteria that form root nodules in the roots of leguminous plants. Generally, leguminous plants symbiose with rhizobia. Rhizobia convert nitrogen in the atmosphere into ammonium nitrogen through the root nodules formed in the roots of leguminous plants and supply it to the host leguminous plants, and soybeans obtain a nitrogen source. Therefore, it is considered that it has been previously known that leguminous plants can symbiose with rhizobia, and if they symbiose with other bacteria in addition to rhizobia, it will be able to hinder the growth promotion of leguminous plants caused by rhizobia. In contrast, the present inventors have found the following new insight: root endophytic plant symbiotic bacteria containing at least one of bacteria of the genus Cladophialophora, bacteria of the genus Exophiala, and bacteria of the genus Veronaeopsis symbiose with rhizobia and plants, do not hinder the growth of leguminous plants, but promote the growth of leguminous plants.

[0037] Examples of rhizobia include bacteria of the genus Bradyrhizobium (e.g., Bradyrhizobium japonicum, etc.), bacterial species of the genus Rhizobium, etc. Among them, as rhizobia, from the viewpoint of further promoting the growth of leguminous plants, Bradyrhizobium japonicum is preferred.

[0038] <Other components> The culture soil for leguminous plants may also contain root endophytic plant symbiotic bacteria, soil, and other components other than rhizobia within the range where the effects of the present invention can be exerted. As other components, for example, solid media (such as amino acids like leucine, methionine, or phenylalanine, or sucrose, etc.) and liquid media (such as water, sterilized water, sterilized distilled water, or physiological saline, etc.), components for stably retaining fungi in the culture soil (such as stabilizers or isotonic agents, etc.), and components for promoting the proliferation of the fungi involved in the present invention (such as malt extract medium (MEB), CM malt yeast medium (CMMY) (a mixture of 8.5 g of CM agar, 15 g of agar, 10 g of malt extract, 1 g of yeast extract, and 1 L of sterilized water), wheat bran, rice bran, or leaf mold, etc.) can be cited.

[0039] 《Cultivation Kit for Leguminous Plants》 The cultivation kit of the present invention is a cultivation kit for leguminous plants containing the culture soil for leguminous plants of the present invention and leguminous plant bodies. According to the present invention, a cultivation kit for leguminous plants that can promote the growth of leguminous plants can be obtained.

[0040] As leguminous plant bodies, for example, seeds, seedlings, etc. of leguminous plants can be cited. The seeds of leguminous plants include not only the state of the seeds before germination but also the seeds with radicles and plumules emerging as a concept. Examples of the seedlings of leguminous plants include seedlings with only cotyledons emerging, seedlings at the three-leaf stage, seedlings at a stage above the three-leaf stage, seedlings proliferated from the parent plant, generation-spanning seedlings (such as seedlings of runners), and cloned seedlings proliferated by grafting, cutting, etc.

[0041] 《Cultivation Method for Leguminous Plants》 The cultivation method for leguminous plants of the present invention includes: cultivating leguminous plants using the culture soil for leguminous plants of the present invention (hereinafter, also referred to as "cultivation process"). According to the present invention, leguminous plants with promoted growth can be cultivated.

[0042] In the cultivation process, from the viewpoint of further promoting the growth of leguminous plants, it is preferable to cultivate leguminous plants by mixing root endophytic plant symbiotic bacteria and rhizobia with the plant body simultaneously. That is, the culture soil for leguminous plants of the present invention preferably further contains rhizobia from the viewpoint of further promoting the growth of leguminous plants.

[0043] The cultivation process can be, for example: after growing leguminous plants from seeds to seedlings using the culture soil for leguminous plants of the present invention, transplanting the seedlings in the soil and cultivating the leguminous plants until the harvest period.

[0044] The cultivation method is not particularly limited as long as it uses the cultivation soil for leguminous plants of the present invention. It can be cultivated solely with the cultivation soil for leguminous plants of the present invention, or the cultivation soil for leguminous plants of the present invention can be mixed with the soil for planting and other cultivation soils for cultivation. When cultivating by mixing the above-mentioned cultivation soil for leguminous plants of the present invention with other soils or cultivation soils, the timing of mixing the cultivation soil for leguminous plants of the present invention can be any one of the time of sowing the seeds of leguminous plants in the soil, the time of transplanting the seedlings at the three-leaf stage in the soil, or the time of planting the seedlings in the soil. Or, it can be the method of sowing the seeds of leguminous plants, transplanting the seedlings at the three-leaf stage, or planting the seedlings in the soil in the soil that has been pre-mixed with the cultivation soil for leguminous plants of the present invention.

[0045] The cultivation conditions can be appropriately designed according to the soil used, the variety of leguminous plants, the weather, etc. For example, the cultivation process can be: after forming flower buds under low-temperature and short-day conditions, cultivating under high-temperature and long-day conditions. The low-temperature and short-day conditions refer to: the temperature exceeds 5°C and is below 15°C, and the light length is above 0 hours and below 12 hours. The high-temperature and long-day conditions refer to: within the range where the temperature exceeds 15°C and is below 25°C (preferably exceeds 20°C and is below 25°C, more preferably 23±1°C), and the light length is within the range of exceeding 12 hours and below 24 hours (preferably exceeds 14 hours and below 24 hours, more preferably above 15 hours and below 17 hours).

[0046] In the cultivation process, for example, the artificial climate chamber for the growth of leguminous plants can be maintained at a constant temperature, and the growth location can be rotated every few days to ensure the uniformity of the temperature.

[0047] Seedling of Leguminous Plant with Cultivation Soil The seedling of a leguminous plant with cultivation soil according to the present invention comprises the cultivation soil for leguminous plants of the present invention and the seedling of a leguminous plant. According to the present invention, a seedling of a leguminous plant with promoted growth can be obtained.

[0048] Use of Cultivation Soil for Leguminous Plants According to the present invention, for cultivating leguminous plants, by using the cultivation soil for leguminous plants of the present invention, the growth of leguminous plants can be promoted. [Examples]

[0049] Hereinafter, the present invention will be described more specifically by way of examples. As long as the present invention does not exceed its gist, it is not limited to the following examples. It should be noted that "parts" are based on mass unless otherwise specified.

[0050] Preparation of Root Endophytic Bacterial Plant Symbiotic Bacteria As root endophytic bacterial plant symbiotic bacteria, fungi of the genus Cladophialophora, namely Cladophialophora chaetospira SK51 (hereinafter also referred to as Cc), fungi of the genus Exophiala, namely Exophiala sp. SK47 (hereinafter also referred to as Esp), and fungi of the genus Veronaeopsis, namely Veronaeopsis simplex Y34 (hereinafter also referred to as Vs) were prepared respectively. The Esp is a fungus deposited with the National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center, located at 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, with an accession number of NITE BP-03540 (date of deposit: November 07, 2022). The Cc is a fungus deposited with the National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center, located at 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, with an accession number of NITE BP-03539 (date of deposit: November 07, 2022). The Vs is a fungus deposited with the National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center, located at 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, with an accession number of NITE BP-01933 (date of deposit: October 11, 2023). These three types of fungi were cultured in 500 mL flasks each containing 250 mL of 2% (by mass) malt extract medium (MEB) under shaking conditions of 23°C and 120 rpm for 4 weeks. After culturing, the three types of mycelia were collected by filtering the culture broth, and the mycelia were washed with sterilized distilled water until the liquid containing the collected mycelia became transparent, so that there was no contamination from the MEB medium. Then, to prevent contamination, a stirrer and laminar flow were used to mix each obtained mycelium and sterilized distilled water at the lowest speed for 1 minute to obtain a pre-culture broth. The survival rate of each fungus in the pre-culture broth was measured by directly plating the pre-culture broth containing the mycelium on 50% (by mass) CMMY agar medium and then culturing at 23°C for 7 days.

[0051] It should be noted that the large-scale production of each fungus was carried out by the following method. Specifically, 10 mL of the pre-culture broth of each fungus (1×10 6The pre-culture solutions of the respective fungi (in hyphal fragments / ml) were added to sterilized plastic bags containing sterilized culture materials (a mixture of 100 g of wheat bran, 100 g of rice bran, 300 g of leaf mold, and 340 mL of sterilized water). Then, the mixtures of the pre-culture solutions of the respective fungi and the culture materials were cultured in a chamber for 3 to 4 weeks to separately prepare materials containing the respective fungi.

[0052] "Preparation of Soil" As the organic cultivation soil, organic soil (manufactured by Sakata Seed Corporation) was prepared.

[0053] The prepared organic cultivation soil had a pH of 6.0 and was used as the "neutral organic cultivation soil". On the other hand, peat moss (manufactured by Kato Kowa Noen) was appropriately mixed into the prepared organic cultivation soil to prepare a "weakly acidic organic cultivation soil" with a pH of 4.0.

[0054] It should be noted that the pH of the soil was measured by the following method. Specifically, for the pH of the soil, the soil and distilled water were mixed at a ratio of 1:2.5 (1:5 when using soil with a high organic matter content), stirred with a reciprocating shaker for more than 1 hour, and then the pH of the suspension was measured by the glass electrode method at 23°C ± 2°C.

[0055] Any of the above-mentioned soils was autoclaved twice under the conditions of 121°C and 30 minutes.

[0056] "Preparation of Culture Soil for Leguminous Plants" The soil with the pH shown in Table 1, the material of the root endophytic bacterial plant symbiotic bacteria of the type shown in Table 1, and rhizobia were mixed to obtain the culture soil for leguminous plants in each example. The root endophytic bacterial plant symbiotic bacteria were mixed so as to be 10% by mass based on the whole culture soil.

[0057] [Table 1] pH of the soil Species of endophytic plant symbiotic bacteria in roots Presence or absence of rhizobia Comparative Example 1 6.0 - Yes Example 1 6.0 Esp Yes Example 2 6.0 Vs Yes Example 3 6.0 Cc Yes Comparative Example 2 4.0 - Yes Example 4 4.0 Vs Yes Example 5 4.0 Cc Yes

[0058] "Cultivation of Soybeans: Examples 1 to 5" Seeds of the soybeans to be cultivated were prepared. The surface of the seeds was sterilized by the method shown below. Specifically, the seeds were immersed in a 70% by mass ethanol solution for 40 seconds and then in a sodium hypochlorite (1% available chlorine) solution for 15 seconds. Then, the seeds were washed 3 times with sterilized distilled water, dried overnight, placed on a 1% by mass water agar medium, and left standing at 23°C for 2 days to germinate. Then, they were grown at 23°C in an artificial climate chamber until the three-leaf stage. It should be noted that in Example 1, seeds of "Fukuyutaka", which have excellent processing characteristics in products such as tofu, were prepared as the soybeans to be cultivated. In Examples 2 to 5, seeds of "Suzumaru", which have excellent processing characteristics in products such as natto, were prepared as the soybeans to be cultivated.

[0059] In a pot with a diameter of 6 cm, materials containing plant symbiotic bacteria of the root endophyte system of the types shown in Table 1, rhizobia, and organic cultivation soil with the pH shown in Table 1 were mixed respectively to obtain culture soil for leguminous plants. The seedlings at the three-leaf stage were planted in this pot, and in an artificial climate chamber, at 30 °C for 14 hours in the bright place and at 22 °C for 10 hours in the dark place, the photosynthetic photon flux density (PPFD) was set to 89.46 m -2 ·s -1 , and they were planted and cultivated. During the cultivation process, water was poured once a day.

[0060] "Cultivation of Soybeans: Comparative Examples 1 - 2" The organic cultivation soil did not contain materials containing plant symbiotic bacteria of the root endophyte system and had the pH shown in Table 1. Other than that, culture soil for leguminous plants was obtained by the same method as in Example 1. Then, soybeans were cultivated by the same method as in Example 1.

[0061] "Evaluation" <Growth State of Soybean Seedlings: 14th Day of Cultivation> Photos showing the (A) overall growth state of the soybean seedlings on the 14th day of cultivation starting from mixing the plant symbiotic bacteria of the root endophyte system with the soybean seedlings in Examples 1, 2, 3 and Comparative Example 1 are shown in Figure 1A . Graphs showing the dry mass of the (B) stem - leaf region and root region of the soybean seedlings on the 14th day of cultivation starting from mixing the plant symbiotic bacteria of the root endophyte system with the soybean seedlings in Examples 1, 2, 3 and Comparative Example 1 are shown in Figure 1B . It should be noted that the dry mass is the value obtained by taking out the soybean plant from the pot after cultivation in the culture soil for leguminous plants in each example, classifying it into the stem - leaf region and root region, and drying it at 40 °C for 72 hours and then measuring the mass. Statistical analysis was performed using SPSS version 20.0 (SPSS, IBM, Armonk, NY, United States), and one - way analysis of variance (ANOVA) and Tukey test (p < 0.05) were carried out. As Figure 1A and Figure 1BAs shown, it can be seen that the soybean seedlings of Examples 1 to 3 symbiotic with the root endophytic plant symbiotic bacteria have a significantly higher dry mass in the stem-leaf region of the seedlings compared to the soybean seedlings of Comparative Example 1 that are not symbiotic with the root endophytic plant symbiotic bacteria, and can promote the growth of leguminous plants. As Figure 1B shown, it can be seen that among the soybean seedlings of Examples 1 to 3 symbiotic with the root endophytic plant symbiotic bacteria, in Examples 2 and 3 where the root endophytic plant symbiotic bacteria are Vs and Cc respectively, compared to Example 1 where the root endophytic plant symbiotic bacteria are Esp, the dry mass in the stem-leaf region of the seedlings is further increased, and the growth promotion of soybean, a leguminous plant, is more excellent.

[0062] <Soybean yield: 140 days after cultivation> The graphs related to the dry mass of soybeans harvested 140 days after cultivation starting from mixing the root endophytic plant symbiotic bacteria with soybean seedlings in Examples 1, 2, 3 and Comparative Example 1 are shown in Figure 2 . It should be noted that the dry yield of the harvested soybeans refers to the dry yield of the soybeans taken out from the pods. The dry yield is measured and statistically analyzed in the same way as in the evaluation of <Growth state of soybean seedlings: 14 days after cultivation>. Figure 2 The "-" marked in the bar graph in [ ] refers to the median (second quartile) of each data. Figure 2 The "×" marked in the bar graph in [ ] refers to the average value of each data. Figure 2 The "*" marked in the bar graph in [ ] refers to the data showing statistical significance as a result of statistical processing. As Figure 2 shown, it can be seen that the soybean plants of Examples 1 to 3 symbiotic with the root endophytic plant symbiotic bacteria have the growth of soybean, a leguminous plant, promoted and the yield increased compared to the soybean plants of Comparative Example 1 that are not symbiotic with the root endophytic plant symbiotic bacteria.

[0063] <Mass of root nodules produced in soybean roots: 40 days after cultivation> The state of root nodules attached to the roots of the plants 40 days after cultivation starting from mixing the root endophytic plant symbiotic bacteria and soybean seedlings in Examples 1, 2, 3 and Comparative Example 1 is shown in Figure 3 . As Figure 3As shown, it can be seen that the soybean plants of Examples 1 to 3 symbiotic with the root endophytic plant symbiotic bacteria have a tendency to have more root nodules formed on the roots compared to the soybean plants of Comparative Example 1 that are not symbiotic with the root endophytic plant symbiotic bacteria. Generally, root nodules tend to supply nutrient sources such as nitrogen to the roots of leguminous plants. Therefore, it is considered that the increase in the amount of root nodules is one of the factors contributing to the increase in the yield and the promotion of growth of the soybeans obtained in each example.

[0064] <Measurement of the amount of phosphoric acid in the soil during cultivation> The graphs related to the phosphoric acid uptake of the soybean plants in Examples 1, 2, 3 and Comparative Example 1 at 0 days, 100 days, and 140 days from the start of cultivation after mixing the root endophytic plant symbiotic bacteria and soybean seedlings are shown in Figure 4 . The phosphoric acid uptake is a value quantitatively obtained by a spectrophotometer measuring device (SPCA-6210, manufactured by Shimadzu Corporation) based on the available phosphoric acid extracted from the soybean seedlings at each cultivation day according to the Bray-I method. In addition, the same statistical analysis as in the evaluation <Growth state of soybean seedlings: 14th day of cultivation> is performed. As Figure 4 shown, it can be seen that the soybean plants of Examples 1 to 3 symbiotic with the root endophytic plant symbiotic bacteria have a tendency to have more phosphoric acid uptake compared to the soybean plants of Comparative Example 1 that are not symbiotic with the root endophytic plant symbiotic bacteria. Generally, phosphoric acid acts as a nutrient source in leguminous plants. Therefore, the increase in phosphoric acid uptake is one of the factors contributing to the increase in the yield and the promotion of growth of the soybeans obtained in each example.

[0065] <Correlation evaluation between the pH of the soil and the growth state of soybeans> The graphs related to the (A) number of leaves of the soybean plants in Examples 4, 5 and Comparative Example 2 at 20 days from the start of cultivation after mixing the root endophytic plant symbiotic bacteria and soybean seedlings are shown in Figure 5A . The graphs related to the (B) dry weight of the soybean plants in Examples 4, 5 and Comparative Example 2 at 20 days from the start of cultivation after mixing the root endophytic plant symbiotic bacteria and soybean seedlings are shown in Figure 5B . The photos showing the (C) growth state of the seedlings of the soybean plants in Examples 4, 5 and Comparative Example 2 at 20 days from the start of cultivation after mixing the root endophytic plant symbiotic bacteria and soybean seedlings are shown in Figure 5C . Figure 5BIn this context, "SDM" refers to Shoot Dry Mass (i.e., the dry weight of leaves and stems (aboveground part)), and "RDM" refers to Root Dry Mass (i.e., the dry weight of roots) (unit: g). The dry weight and statistical analysis were carried out by the same method as in evaluating <Growth state of soybean seedlings: 14th day of cultivation>. As Figure 5A shown in ~C, the soybean plants in Example 4 with the root endophytic plant symbiotic bacterium Vs, compared with the soybean plants in Example 5 with the root endophytic plant symbiotic bacterium Cc and the soybean plants in Comparative Example 2 that were not symbiotic with the root endophytic plant symbiotic bacterium, showed an increasing trend in the number of leaves and dry mass. That is, it was found that even in a weakly acidic soil environment with a pH of 4, the growth of leguminous plants can be promoted.

[0066] <Cultivation of Soybeans: Example 6> The soybean seeds were sterilized using the same materials and methods as in Example 1. Then, the seeds were placed in a culture soil for leguminous plants containing the types of root endophytic plant symbiotic bacteria shown in Table 2, and left to germinate and grow at 23°C for 7 days in an artificial climate chamber. Then, on the 7th day of cultivation, the seedlings were transplanted using a culture soil containing rhizobia and cultivated in an artificial climate chamber at 30°C for 14 hours in the light and 22°C for 10 hours in the dark, with the photosynthetic photon flux density (PPFD) set at 89.46 m -2 ·s -1 , and transplanted and cultivated. During cultivation, water was applied once a day.

[0067] <Cultivation of Soybeans: Comparative Example 3> The culture soil for leguminous plants containing the root endophytic plant symbiotic bacteria was set as a culture soil without the root endophytic plant symbiotic bacteria, and except for this, soybeans were cultivated by the same method as in Example 6.

[0068] <Cultivation of Soybeans: Example 7> The surface of the soybean seeds was sterilized using the same materials and methods as in Example 1. Then, the seeds were placed in a culture soil for leguminous plants containing the types of root endophytic plant symbiotic bacteria and rhizobia shown in Table 2, and left to germinate and grow at 23°C for 7 days in an artificial climate chamber. Then, on the 4th day of cultivation, the seedlings were cultivated in an artificial climate chamber at 30°C for 14 hours in the light and 22°C for 10 hours in the dark, with the photosynthetic photon flux density (PPFD) set at 89.46 m -2 ·s -1 , and transplanted and cultivated. During cultivation, water was applied once a day.

[0069] "Cultivation of Soybeans: Comparative Example 4" The culture soil for leguminous plants containing root endophytic plant symbiotic bacteria and rhizobia was made into a culture soil containing only rhizobia without root endophytic plant symbiotic bacteria. Except for this, soybeans were cultivated by the same method as in Example 7.

[0070] [Table 2]

[0071] "Evaluation" <Evaluation of the timing of combination of rhizobia and root endophytic plant symbiotic bacteria> The graphs related to the number of (A) roots of the soybean plants on the 27th day of cultivation in Example 6 and Comparative Example 3 are shown in Figure 6A . The graphs related to the number of (B) leaves of the soybean plants on the 27th day of cultivation in Example 6 and Comparative Example 3 are shown in Figure 6B . The graphs related to the number of (A) roots of the soybean plants on the 20th day of cultivation in Example 7 and Comparative Example 4 are shown in Figure 7A . The graphs related to the number of (B) leaves of the soybean plants on the 20th day of cultivation in Example 7 and Comparative Example 4 are shown in Figure 7B . As shown in Figure 6A , B and Figure 7A , B, it can be seen that regardless of the timing of mixing the culture soil for leguminous plants containing root endophytic plant symbiotic bacteria with the soybean plants, the culture soil for leguminous plants in the examples can promote the growth of soybeans as leguminous plants compared with the culture soil for leguminous plants in the comparative examples. In addition, as shown in Figure 6A and Figure 7A , it can be seen that mixing symbiotic bacteria and rhizobia with seeds at the same cultivation days (i.e., the same timing) can further promote the growth of soybeans as leguminous plants

[0072] The content of Japanese Patent Application No. 2022-181348 filed on November 11, 2022 is incorporated herein by reference in its entirety. All documents, patent applications, and technical standards described in this specification are incorporated herein by reference to the same extent as when each document, patent application, and technical standard is specifically and separately described.

Claims

1. A culture soil for leguminous plants, wherein, Comprising a root endophytic plant symbiotic bacterium and soil, wherein the root endophytic plant symbiotic bacterium comprises at least one bacterium selected from the group consisting of fungi of the genus Cladophialophora, fungi of the genus Exophiala, and fungi of the genus Veronaeopsis.

2. The culture soil for leguminous plants according to claim 1, wherein, The culture soil for leguminous plants further comprises rhizobia.

3. The culture soil for leguminous plants for plants according to claim 1, wherein, The fungus of the genus Cladophialophora is Cladophialophora chaetospira.

4. The culture soil for leguminous plants according to claim 1, wherein, The root endophytic plant symbiotic bacterium is a fungus of the genus Veronaeopsis.

5. The culture soil for leguminous plants according to claim 4, wherein, The pH of the soil is 4 or more and less than 6.

6. The culture soil for leguminous plants according to claim 1, wherein, The root endophytic plant symbiotic bacterium is a fungus of the genus Cladophialophora.

7. The culture soil for leguminous plants according to claim 6, wherein, The pH of the soil is 6 or more and 7 or less.

8. A cultivation kit for leguminous plants, wherein, Comprising the culture soil for leguminous plants according to any one of claims 1 to 7, and a leguminous plant body.

9. A cultivation method for a leguminous plant, wherein, Comprising cultivating a leguminous plant using the culture soil for leguminous plants according to claim 1.

10. The cultivation method of leguminous plants according to claim 9, wherein, In the cultivation, the root endophytic plant symbiotic bacterium and rhizobia are simultaneously mixed with the plant body to cultivate the leguminous plant.

11. A seedling of a leguminous plant with cultivation soil, wherein, Comprising the culture soil for leguminous plants according to any one of claims 1 to 7, and a seedling of a leguminous plant.

12. Use of the culture soil for leguminous plants according to any one of claims 1 to 7 in the cultivation of leguminous plants.

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